Hydroxyl radical scavenging action of capsaicin

We recently reported that capsaicin (CAP) is capable of scavenging peroxyl radicals derived from 2,2'-azobis(2,4-dimethylvaleronitrile) as measured by electron spin resonance (ESR) spectroscopy. The present study describes the hydroxyl radical (HO*) scavenging ability of CAP as measured by DNA strand scission assay and by an ESR spin trapping technique with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). The Fenton reaction [Fe(II)+ H(2)O(2) --> Fe(III) + HO* + HO(-)] was used as a source of HO*. The incubation of DNA with a mixture of FeSO(4) and H(2)O(2) caused DNA strand scission. The addition of CAP to the incubation mixture decreased the strand scission in a concentration-dependent manner. To understand the antioxidative mechanism of CAP, we used an ESR spin trapping technique. Kinetic competition studies using different concentrations of DMPO indicated that the decrease of the oxidative DNA damage was mainly due to the scavenging of HO* by CAP, not to the inhibition of the HO* generation system itself. We estimated the second order rate constants in the reaction of CAP and common HO* scavengers with HO* by kinetic competition studies. By comparison with the common HO* scavengers, CAP was found to scavenge HO* more effectively than mannitol, deoxyribose and ethanol, and to be equivalent to DMSO and benzoic acid, demonstrating that CAP is a potent HO* scavenger. The results suggest that CAP may act as an effective HO* scavenger as well as a peroxyl radical scavenger in biological systems.     

In vitro scavenging activity for reactive oxygen species by N-substituted indole-2-carboxylic acid esters.

The hydroxyl radical (HO*)- and superoxide anion radical (O* (2))-scavenging activity, as well as the singlet oxygen ((1)O(2))-quenching property of N-substituted indole-2-carboxylic acid esters (INDs) were investigated by deoxyribose degradation assay, a chemiluminescence method and the electron spin resonance (ESR) spin-trapping technique. This novel group of compounds was developed as a search for cyclooxygenase-2 (COX-2)-selective enzyme inhibitors. The results obtained demonstrated that of the 16 compounds examined, five inhibited light emission from the superoxide anion radical (O* (2))-DMSO system by at least 60% at a concentration of 1 mmol/L, nine prevented the degradation of deoxyribose induced by the Fenton reaction system (range 3-78%) or scavenged hydroxyl radicals (HO*) directly (range 8-93%) and 14 showed the (1)O(2)-quenching effect (range 10-74%). These results indicate that majority of the indole esters tested possess the ability to scavenge O(-) (2) and HO radicals and to quench (1)O(2) directly, and consequently may be considered effective antioxidative agents.     

Prooxidant and antioxidant action of 4-(4-phenoxybenzoyl)benzoic acid derivatives

4-(4-Phenoxybenzoyl)benzoic acid derivatives (PBADs) were found to inhibit rat and human alpha-reductase isozymes 1 and 2 in vitro. Chemiluminescence (CL), electron spin resonance, spin trapping techniques, and spectrophotometry were used to examine the effect of PBADs on reactive oxygen species (superoxide radical, O(2)(.-); hydroxyl radical, HO(*); singlet oxygen, (1)O(2)) generating systems. All test compounds at a concentration of 0.5 mM enhanced the CL from O(2)(.-) up to fivefold, which was recorded as the light sums during 1 min. At 0.38 mM PBAD enhanced production of HO(*) from H(2)O(2) in the presence of Co(II) up to 90%, as measured by a deoxyribose assay. Using the spin trap agent 5,5-dimethyl-1-pyrroline-N-oxide, it was found that the amplitude of the signal arising from the Fenton-like reaction [Co(II)/H(2)O(2)] was significantly diminished by the test compounds. The compounds also inhibited the (1)O(2) dependent 2,2,6,6-tetramethylpiperidine-N-oxide radical, which is generated in the acetonitrile/H(2)O(2) system. The measured rate constants of (1)O(2)-dimol quenching by PBAD were in the range of (0.8-2.6) x 10(8) M(-1) s(-1). The interaction between PBAD and (1)O(2) was also checked using a spectrophotometry method based on bleaching of p-nitrosodimethylaniline. These results indicate that PBAD may directly scavenge HO(*) and (1)O(2), but not O(2)(.-). However, the compounds that were examined had prooxidant ability under some reaction conditions.     

Comparison of the effects of O2*-/HO* free radical- and copper ions-oxidized LDL or lipoprotein(a) on the endothelial cell releases of tissue plasminogen activator and plasminogen activator inhibitor-1.

We investigated the effects of low density-lipoproteins (LDL) and lipoprotein(a) [Lp(a)] oxidized by O2*-/HO* free radicals generated by gamma radiolysis of water, on the release of tissue Plasminogen Activator (tPA) and of its main inhibitor Plaminogen Activator Inhibitor-1 (PAI-1) by human umbilical vein endothelial cells (HUVEC). These effects were compared to those of lipoproteins issued from the same preparations but oxidized by the classical copper ions procedure. The results showed that O2*-/HO* free radical oxidized LDL and Lp(a) led to a dramatic decrease of PAI-1 release but did not affect tPA release, whereas copper oxidation of lipoproteins resulted in an increase in PAI-1 release and a decrease in tPA release. Chemical analysis revealed that O2*-/HO* free radical oxidized lipoproteins exhibited very much lower levels of phosphatidylcholine hydroperoxides, lysophosphatidylcholine and oxysterols (7-ketocholesterol, 7beta-hydroxycholesterol, 5,6beta-epoxycholesterol) than copper oxidized LDL. Thus, the discordant effects of O2*-/HO* oxidized and copper oxidized LDL and Lp(a) on the endothelial releases of PAI-1 and tPA appeared to be due to qualitatively and/or quantitatively different formation of oxidized components by the two oxidation processes.     

Anti-inflammatory and antioxidant activity of a medicinal tincture from Pedilanthus tithymaloides

Pedilanthus tithymaloides (L.) Poit. (Euphorbiaceae) is a low tropical American shrub with a reported wide range of healing properties such as emetic, anti-inflammatory, antibiotic, antiseptic, antihemorrhagic, antiviral, antitumoral, and abortive. In the present study, a tincture from P. tithymaloides collected in Cuba was evaluated for its in vivo anti-inflammatory activity, using the rat paw oedema assay, and for its in vitro scavenging effects on reactive oxygen species (ROS) (HO*, O2*-, HOCl, ROO* and H2O2), reactive nitrogen species (RNS) (ONOO- and *NO), and DPPH* radical. The protein, free amino acid, and phenolic contents of the tincture were also determined. Pertaining to the anti-inflammatory activity, the intraperitoneal administration of the tincture inhibited carrageenan-induced rat paw oedema, whereas in the scavenging assays the tincture showed to be effective against all the assayed ROS and RNS, specially for HO* (IC50 = 345+/-77 microg/mL), O2*- (IC50 = 143+/-7 microg/mL), HOCl (IC50 = 113+/-20 microg/mL), ONOO- (IC50 = 44+/-3 microg/mL), and *NO (IC50 = 54+/-4 microg/mL), but displayed weak activity in the DPPH* assay. The protein content of the tincture was 0.70%, and twenty free amino acids were identified and quantified. The content of total phenolics was 17.4+/-0.15 mg of gallic acid equivalents (GAE)/g dry material. These results provide scientific support for the empirical use of P. tithymaloides tincture as an anti-inflammatory medicine.     

The differential effects of superoxide anion, hydrogen peroxide and hydroxyl radical on cardiac mitochondrial oxidative phosphorylation

The involvement of reactive oxygen species (ROS) in cardiac ischemia-reperfusion injuries is well-established, but the deleterious effects of hydrogen peroxide (H(2)O(2)), hydroxyl radical (HO*) or superoxide anion (O(2)*(-) ) on mitochondrial function are poorly understood. Here, we report that incubation of rat heart mitochondria with each of these three species resulted in a decline of the ADP-stimulated respiratory rate but not substrate-dependent respiration. These three species reduced oxygen consumption induced by an uncoupler without alteration of the respiratory chain complexes, but did not modify mitochondrial membrane permeability. HO* slightly decreased F1F0-ATPase activity and HO* and O(2)*(-) partially inhibited the activity of adenine nucleotide translocase; H(2)O(2) failed to alter these targets. They inhibited NADH production by acting specifically on aconitase for O(2)*(-) and alpha-ketoglutarate dehydrogenase for H(2)O(2) and HO*. Our results show that O(2)*(-), H(2)O(2) and HO* act on different mitochondrial targets to alter ATP synthesis, mostly through inhibition of NADH production.     

The Haber-Weiss cycle--70 years later

The chain reactions HO* + H2O2 --> H2O + O2*- + H+ and O2*- + H+ + H2O2 --> O2 + HO* + H2O, commonly known as the Haber-Weiss cycle, were first mentioned by Haber and Willst?tter in 1931. George showed in 1947 that the second reaction is insignificant in comparison to the fast dismutation of superoxide, and this finding appears to have been accepted by Weiss in 1949. In 1970, the Haber-Weiss reaction was revived by Beauchamp and Fridovich to explain the toxicity of superoxide. During the 1970s various groups determined that the rate constant for this reaction is of the order of 1 M(-1) s(-1) or less, which confirmed George's conclusion. The reaction of superoxide with hydrogen peroxide was dropped from the scheme of oxygen toxicity, and superoxide became the source of hydrogen peroxide, which yields hydroxyl radicals via the Fenton reaction, Fe2+ + H2O2 --> Fe3+ + HO- + HO*. In 1994, Kahn and Kasha resurrected the Haber-Weiss reaction again, but this time the oxygen was believed to be in the singlet (1delta(g)) state. As toxicity arises not from a Fenton-catalysed Haber-Weiss reaction, but from the Fenton reaction, the Haber-Weiss reaction should not be mentioned anymore.     

Antioxidants suitable for use with chemiluminescence to identify oxyradical species

From a panel of 24 alleged antioxidants the most suitable antioxidants (AO) for use with chemiluminescence (CL) experiments were determined. Superoxide dismutase (SOD), using luminol as the chemiluminescence probe (Lum-CL), was inhibitory only towards O2*- and not HO* or (1)O2. SOD was thus a suitable antioxidant for O2*-, as was tiron. Tiron had advantages, however, since SOD acted as a pro-oxidant in the presence of H2O2 or H2O2/HO* generators. The two most suitable antioxidants for (1)O2 were diphenylisobenzofuran (DBF) and tryptophan, for both Lum and Lucigenin-CL (Luc-CL). Desferrioxamine, with both Lum and Luc-CL, was a very effective scavenger for HO*, but appeared to be an even more effective scavenger for (1)O2. Cysteamine showed the best discrimination between IC50s when the two (1)O2 generators NaOCl/H2O2 and NDPO2 were compared. Cysteamine was, therefore, the only scavenger that was appropriate for studies with hypochlorite. Melatonin, with Lum-CL, was found to be the most suitable scavenger for HO*. Mannitol, the classical AO for HO*, was not suitable when used with CL since it acted as a pro-oxidant. Some of the AOs revealed either calyx- or bell-shaped CL inhibition profiles and presumably, therefore, may act as both pro- or antioxidants at different concentrations. Antioxidants showing these kinds of dual activities should be used with caution in CL studies.     

The roles of O2-, HO(.), and secondarily derived radicals in oxidation reactions catalyzed by vanadium salts.

V(IV) decomposed H2O2, with evolution of O2, in a free radical chain process involving O2- and HO(.). When V(IV) was limiting, the presence of V(V) augmented O2 evolution because it allowed production of additional V(IV) from the reduction of V(V) by O2-. Gradual addition of V(IV) increased the yield of O2 evolved, per V(IV) added, to greater than 1--a clear indication of a free radical chain reaction. Reductants such as ethanol, Hepes, and NADH imposed a phase of O2 consumption because of HO.-initiated oxidation reactions. The radical produced from the reaction of HO. with ethanol was unable to directly oxidize NADH, whereas that produced from Hepes was able to do so. Ethanol consequently inhibited the oxidation of NADH by anaerobic V(IV) + H2O2, whereas Hepes did not. These results, and others reported herein, are explained on the basis of a coherent set of reactions. Data already in the literature are also clarified on the basis of these reactions.     

Scavenging of reactive oxygen species by some nonsteroidal anti-inflammatory drugs and fenofibrate

Ketoprofen and tolmetin are widely used nonsteroidal anti-inflammatory drugs, whereas fenofibrate belongs to a family of hypolipidemic drugs used in the prevention of cardiovascular diseases. The aim of this study was to assess effect of these drugs on reactions generating reactive oxygen species (ROS). The following generators of ROS were used: 18-crown-6/KO(2) dissolved in DMSO as a source of superoxide radical (O(.-)(2), the Fenton-like reaction (Cu/H(2)O(2)) for hydroxyl radical (HO(.)), 2,2'-azobis (2-amidino-propane) dichloride (AAPH) as peroxyl radical (ROO(.)) generator, and a mixture of alkaline aqueous H(2)O(2) and acetonitrile for singlet oxygen ((1)O(2)). Measurements were done using chemiluminescence, fluorescence, and spin-trapping with 2,2,6,6-tetramethylpiperidine combined with electron spin resonance spectroscopy (ESR), and a deoxyribose assay based on the spectrophotometry. The results obtained demonstrated that all tested drugs were active against O(.-)(2). There was a clear ranking of drug inhibition effects on chemiluminescence from the O(.-)(2) system: ketoprofen > tolmetin > fenofibrate. The examined compounds inhibited the HO(.)-dependent deoxyribose degradation and scavenged the ROO(.) concentration dependently with an order of potencies similar to that of the superoxide radical system. Hence, these results indicate that the studied drugs show broad ROS scavenging property and, as a consequence, might decrease tissue damage due to the ROS and thus to contribute to anti-inflammatory therapy.     

Manganese complexes of curcumin analogues: evaluation of hydroxyl radical scavenging ability, superoxide dismutase activity and stability towards hydrolysis

In order to improve the antioxidant property of curcumin and its analogue, diacetylcurcumin, manganese was incorporated into the structures in order to enhance superoxide dismutase (SOD) activity. Manganese (Mn) complexes of curcumin (CpCpx) and diacetylcurcumin (AcylCpCpx) were synthesized and firstly investigated for SOD activity and hydroxyl radical (HO*) scavenging ability. SOD activity was evaluated by both the nitroblue tetrazolium (NBT) reduction assay and electron paramagnetic resonance (EPR) with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin trapping agent. CpCpx and AcylCpCpx inhibited the NBT reduction and decreased the DMPO/OOH adduct much greater than corresponding antioxidants or ligands, with IC50 values of 29.9 and 24.7 microM (NBT), and 1.09 and 2.40 mM (EPR), respectively. For EPR, potassium superoxide (KO2) was used as a source of O2- where qualitative results suggested that CpCpx and AcylCpCpx were SOD mimics, which catalyze the conversion of O2- to dioxygen and hydrogen peroxide (H2O2). Additionally, CpCpx and AcylCpCpx exhibited the great inhibition of DMPO/OH adduct formation with an IC50 of 0.57 and 0.37mM, respectively, which were comparable to that of curcumin (IC50 of 0.64 mM), indicating that both Mn complexes are also an effective HO* scavenger. The stability against hydrolysis in water, various buffers and human blood/serum was carried out in vitro. It was found that both Mn complexes were pH and salt concentration dependent, being more stable in basic pH. In the human blood/serum test, CpCpx was more stable against hydrolysis than AcylCpCpx with about 10 and 20% of free Mn2+ releasing, respectively.     

HO2*: the forgotten radical

HO2*, usually termed either hydroperoxyl radical or perhydroxyl radical, is the protonated form of superoxide; the protonation/deprotonation equilibrium exhibits a pK(a) of around 4.8. Consequently, about 0.3% of any superoxide present in the cytosol of a typical cell is in the protonated form. This ratio is rather accurately reflected by the published literature on the two species, as identified by a PubMed search; at the time of writing only 28 articles mention "HO2," "hydroperoxyl" or "perhydroxyl" in their titles, as against 9228 mentioning superoxide. Here it is argued that this correlation is not justifiable: that HO2*'s biological and biomedical importance far exceeds the attention it has received. Several key observations of recent years are reviewed that can be explained much more economically when the participation of HO2* is postulated. It is suggested that a more widespread appreciation of the possible role of HO2* in biological systems would be of considerable benefit to biomedical research.     

Scavenging of reactive oxygen species by the plant phenols genistein and oleuropein.

The plant-derived phenolic compounds genistein and oleuropein are known to exhibit several biological properties, many of which may result from their antioxidant and free radical scavenger activity. In this paper we report the results of a complex study of antioxidant activity of genistein and oleuropein, using electron spin resonance (ESR), chemiluminescence, fluorescence and spectrophotometric techniques. Different reaction systems were applied to study the inhibitory effect of the phenolic compounds studied: (a) the potassium superoxide/18-crown-6 dissolved in DMSO system, which generates superoxide radical (O(2).(-)) and hydrogen peroxide (H(2)O(2)); (b) the Co(II)-EDTA-H(2)O(2) system (the Fenton-like reaction), which generates hydroxyl radical (HO.); (c) 2,2'-azobis(2-amidino-propane)dichloride (AAPH) as the peroxyl radical (ROO.) generator, and the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical test. Results showed that genistein and oleuropein decreased the chemiluminescence sum from the O(2).(-) generating system, an inhibitory effect that was dependent on their concentration. These compounds also reacted with ROO radicals and they showed activity about two-fold greater than the standard Trolox. The antioxidant effects were studied at different concentrations and reflected in protection against the fluorescence decay of beta-phycoerythrin (beta-PE), due to ROO. attack on this protein. Using the Fenton-like reaction and the spin trap agent 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), the phenolic compounds examined were found to inhibit DMPO-.OH radical formation in the range 10-90% at concentrations of 0.1 mmol/L to 2 mmol/L. Furthermore, these compounds also inhibited HO.-dependent deoxyribose degradation; about 20% and 60% inhibitions were observed in the presence of 0.5 mmol/L genistein and oleuropein, respectively. It was also demonstrated that genistein had a weaker DPPH radical scavenging activity than oleuropein. Our results confirm good scavenging activity towards O(2).(-), HO. and ROO. and the antioxidant effect of genistein and oleuropein.     

Mn2+, Co2+, Cu2+ and Zn2+ complexes with two macrocyclic ligands bearing L-lactate-like functions: potentiometric studies and evaluation of superoxide-scavenging properties of the Mn2+ complex

Some aerobic organisms devoid of SOD use Mn2+ chelates to scavenge the O2- radical. Since the Mn2+-bis(lactato)diaquo complex is known as having a high SOD-like activity, we prepared manganese(II) complexes with triazamacrocyclic ligands bearing L-lactate-like functions in order to obtain model compounds able to disproportionate the superoxide radical. Thus, two macrocyclic ligands, N,N',N"-tris[2(S)-hydroxybutyric acid]-1,4,7-triazacyclononane, L1, and N,N',N"-tris[2(S)-hydroxybutyric acid]-1,5,9-triazacyclododecane, L2, were prepared and their capacity to retain the Mn2+ ion in aqueous solution was determined from potentiometric experiments. The chelating properties in aqueous solution of each ligand towards Co2+, Cu2+ and Zn2+ ions were also determined. L1 forms complexes with Mn2+, Co2+, Cu2+ and Zn2+ ions with stability constants of 8.33(5), 15.78(5), 17.65(3) and 14.32(1), respectively. L2 forms complexes with Cu2+ and Zn2+ ions with stability constants of 10.67(1) and 6.98(3), respectively. But the constants related to the Mn2+ and Co2+ complexes were too low to be determined by the method used. The stability constants values calculated for L2 complexes are significantly lower than those for the corresponding complexes of L1. Additional spectroscopic measurements were carried out on the Mn2+-L1 system. The electronic spectrum of this system showed a pH-dependence that may be consistent with the formation of hydroxo-species as the ESR spectra recorded at 120 K did not show oxidation of the Mn2+ ion in the pH range studied. The superoxide-scavenging activity of the manganese(II)-L1 complex was investigated using the cytochrome c assay. The Mn2+-L1 system showed an IC50 value of 1.7 microM which indicates that it appears as a potent SOD mimic.     

Gamma radiolysis as a tool to study lipoprotein oxidation mechanisms

Well-defined quantities of *OH, O2*-,HO2* or RO2*)radicals (reactive oxygen species) can be specifically produced by radiolysis of water or ethanol. Such radical species can initiate one-electron oxidation or one-electron reduction reactions on numerous biological systems. The oxidative hypothesis of atherosclerosis classically admits the involvement of the oxidation of low density lipoproteins (LDLs) but also of high density lipoproteins (HDLs) in the development of the atherosclerotic process. The initiation mechanisms of this oxidation are still incompletely defined, although free radicals are likely involved. Therefore, gamma-radiolysis appears as a method of choice for the in vitro study of the mechanisms of oxidation of LDLs and HDLs by oxygen-centred free radicals (*OH, O2*-,HO2* and RO2*). Radiolytically oxidized lipoproteins exhibited a very well defined oxidation status (radiation dose-dependent quantification of vitamin E, beta-carotene, lipid peroxidation, protein carbonylation ...). gamma-Radiolysis is a less drastic method than other oxidation procedures such as for example copper ions. Moreover, gamma-radiolysis is also especially suitable for studying the reducing properties of antioxidant compounds with regard to their scavenging capacity.     

Trapping of free radicals with direct in vivo EPR detection: a comparison of 5,5-dimethyl-1-pyrroline-N-oxide and 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide as spin traps for HO* and SO4*-.

To spin trap hydroxyl radical (HO*) with in vivo detection of the resultant radical adducts, the use of two spin traps, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO) (10 mmol/kg) has been compared. In mice treatment with 5-aminolevulinic acid and Fe3+ resulted in detection of adducts of hydroxyl radicals (HO*), but only with use of DEPMPO. Similarly, 'HO* adducts' generated via nucleophilic substitution of SO4*- adducts formed in vivo could be observed only when using DEPMPO as the spin trap. The reasons for the differences observed between DEPMPO and DMPO are likely due to different in vivo lifetimes of their hydroxyl radical adducts. These results seem to be the first direct in vivo EPR detection of hydroxyl radical adducts.     

Evaluation of N-hydroxy-2-thiopyridone as a nonmetal dependent source of the hydroxyl radical (HO.) in aqueous systems.

N-hydroxy-2-thiopyridone (1), an established source of the hydroxyl radical (HO., Boivin, J., Crepon, E., and Zard, S. Z. (1990) Tetrahedron Lett. 31, 6869-6872), produced HO. under conditions directly applicable to biological studies. Generation of HO. by subjecting 1 to irradiation with visible light was monitored in the following "HO." assays: deoxyribose degradation, addition to dimethyl sulfoxide, and hydroxylation of salicylate and phenol. All four assays demonstrated the production of HO. from 1 (added as a sodium salt) under mild conditions in aqueous buffer systems. An improved analysis method was developed for the phenol assay. A time course analysis demonstrated that a flux of HO. is generated from 1 throughout the irradiation period, in contrast to the classical Fenton reaction of H2O2 with a transition metal in which a burst of HO. is generated in a short time period. While a thiyl radical is generated from 1 concurrent with HO. generation, this species does not contribute to, or interfere with, any of the HO. assays, suggesting that it is weakly reactive in aqueous buffers. Thus, irradiation of 1 can be used as an alternative, complementary, approach for the unequivocal generation of the biologically significant and reactive HO..     

Generation of *OH initiated by interaction of Fe2+ and Cu+ with dioxygen; comparison with the Fenton chemistry

Iron and copper toxicity has been presumed to involve the formation of hydroxyl radical (*OH) from H2O2 in the Fenton reaction. The aim of this study was to verify that Fe2+-O2 and Cu+-O2 chemistry is capable of generating *OH in the quasi physiological environment of Krebs-Henseleit buffer (KH), and to compare the ability of the Fe2+-O2 system and of the Fenton system (Fe2+ + H2O2) to produce *OH. The addition of Fe2+ and Cu+ (0-20 microM) to KH resulted in a concentration-dependent increase in *OH formation, as measured by the salicylate method. While Fe3+ and Cu2+ (0-20 microM) did not result in *OH formation, these ions mediated significant *OH production in the presence of a number of reducing agents. The *OH yield from the reaction mediated by Fe2+ was increased by exogenous Fe3+ and Cu2+ and was prevented by the deoxygenation of the buffer and reduced by superoxide dismutase, catalase, and desferrioxamine. Addition of 1 microM, 5 microM or 10 microM Fe2+ to a range of H2O2 concentrations (the Fenton system) resulted in a H2O2-concentration-dependent rise in *OH formation. For each Fe2+ concentration tested, the *OH yield doubled when the ratio [H2O2]:[Fe2+] was raised from zero to one. In conclusion: (i) Fe2+-O2 and Cu+-O2 chemistry is capable of promoting *OH generation in the environment of oxygenated KH, in the absence of pre-existing superoxide and/or H2O2, and possibly through a mechanism initiated by the metal autoxidation; (ii) The process is enhanced by contaminating Fe3+ and Cu2+; (iii) In the presence of reducing agents also Fe3+ and Cu2+ promote the *OH formation; (iv) Depending on the actual [H2O2]:[Fe2+] ratio, the efficiency of the Fe2+-O2 chemistry to generate *OH is greater than or, at best, equal to that of the Fe2+-driven Fenton reaction.